1. Field of the Invention
The present invention relates to a method of calibration for improving the accuracy of the process of estimating the volume of a cell clump from an image including the cell clump.
2. Description of the Background Art
A screening which narrows down compounds serving as candidates for medical and pharmaceutical products is performed in the course of research and development of the medical and pharmaceutical products. An example of the screening includes the steps of: preparing a plurality of culture solutions into which biological cells are put; adding compounds to the culture solutions while changing various conditions to cultivate the cells; and narrowing down the compounds serving as candidates for medical and pharmaceutical products, based on the culture states of the cells.
In such a screening, a reagent has been hitherto used for the assessment of the culture states of the cells. Specifically, a reagent is applied to the cells to cause a chemical reaction between specific molecules in the cells and the reagent. The culture states of the cells are judged by absorbance measurement of such optical changes. This method, however, has required the costly reagent, and also has required much time for the chemical reaction. In addition, this method has been incapable of observing changes in the same cell with time because the reagent destroys cell walls.
In recent years, three-dimensional culture such that cells are cultivated in three dimensions has been performed to investigate the effects of medical and pharmaceutical products in an environment closer to that in a living body. An important object to be observed in such three-dimensional culture is the state of a spheroid that is a cell clump comprised of a group of three-dimensionally aggregated cells. However, the use of the absorbance measurement for the observation of such a cell clump gives rise to problems in requiring the costly reagent, in requiring much time for the chemical reaction and in being incapable of observing changes with time, as in the aforementioned cases.
To solve such problems, an attempt has been made in recent years to develop an apparatus for observing the culture states of cell clumps by photographing the cell clumps at a high resolution without using any reagent. This apparatus photographs a well plate having a plurality of depressions or wells for culture at predetermined time intervals to clip images of each of the wells from the resultant photographed images. The culture states of the cell clumps in each well are assessed by displaying the clipped images on a display part and then comparing and analyzing the clipped images.
Conventional apparatuses for performing image processing by acquiring the images of cells are disclosed, for example, in Japanese Patent Application Laid-Open No. 2010-510812, Japanese Patent Application Laid-Open No. 2001-512824, and U.S. Pat. No. 7,718,131.
An important indicator for the assessment of the culture state of a cell clump is the volume of the cell clump. Thus, the development of the technique of estimating the volume of a cell clump from the image of the cell clump has been advanced especially in recent years. An example of the process of estimating the volume of a cell clump includes: converting the optical density of the cell clump in an image into the height thereof; and calculating the volume of the cell clump, based on the height after the conversion and the area of the cell clump.
However, a relationship between the optical density of pixels in the image and the height of the cell clump varies depending on the type and culturing conditions of cells. To determine the volume of a cell clump with accuracy, it is preferable that the relationship between the optical density of the pixels and the height of the cell clump is adjusted by calibration for each object to be observed.